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WO2007022425A2 - Vaccin a sous-unites recombinees du virus de la grippe - Google Patents

Vaccin a sous-unites recombinees du virus de la grippe Download PDF

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Publication number
WO2007022425A2
WO2007022425A2 PCT/US2006/032353 US2006032353W WO2007022425A2 WO 2007022425 A2 WO2007022425 A2 WO 2007022425A2 US 2006032353 W US2006032353 W US 2006032353W WO 2007022425 A2 WO2007022425 A2 WO 2007022425A2
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Prior art keywords
protein
immunogenic composition
influenza
subunit
recombinant
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PCT/US2006/032353
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English (en)
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WO2007022425A9 (fr
WO2007022425A3 (fr
Inventor
Carolyn Weeks-Levy
David E. Clements
Steven A. Ogata
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Hawaii Biotech, Inc.
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Priority to EP06801866A priority Critical patent/EP1945250A4/fr
Priority to CA002656705A priority patent/CA2656705A1/fr
Priority to AU2006279323A priority patent/AU2006279323B2/en
Publication of WO2007022425A2 publication Critical patent/WO2007022425A2/fr
Publication of WO2007022425A9 publication Critical patent/WO2007022425A9/fr
Publication of WO2007022425A3 publication Critical patent/WO2007022425A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/103—Plasmid DNA for invertebrates
    • C12N2800/105—Plasmid DNA for invertebrates for insects

Definitions

  • a sequence listing file in ST.25 format on CD-ROM is appended to this application and fully incorporated herein by reference.
  • the sequence listing information recorded in computer readable form is identical to the written sequence listing (per WIPO ST.25 para. 39, the information recorded on the form is identical to the written sequence listing).
  • the format is ISO 9660; the operating system compatibility is MS- Windows; the single file contained on each CD-ROM is named "FLU.S2.ADJ.04.ST25.txt" and is a text file produced by Patentln 3.3 software; the file size in bytes is 35 KB; and the date of file creation is 16 August 2006.
  • the contents of the two CD-ROMs submitted herewith are identical.
  • the invention relates to vaccine formulations designed to protect against influenza.
  • the vaccine formulations comprise recombinant subunit proteins derived from influenza virus, and optionally include one or more adjuvants.
  • Subunit protein is defined here as any protein derived or expressed independently from the complete organism that it is derived from.
  • a subunit protein may represent a full length native protein sequence or any fraction of the full length native protein sequence.
  • a subunit protein may contain in addition to the full length or partial protein sequence, one or more sequences, which may contain sequences that are homologous or heterologous to the organism from which the primary sequence was derived.
  • subunit protein As a single protein molecule that co-assembles with other protein molecules to form a multimeric or oligomeric protein.
  • the subunit proteins of the invention are produced in a cellular production system by means of recombinant DNA methods and, after purification, are formulated in a vaccine. [04J Each year an estimated 20% of the US population will develop influenza. Approximately 150,000 of those infected will be hospitalized (Schoenbaum, Am, J, Med. (1987) 82(Suppl 6A):26-30; Simonsen et al., Arcklntern. Med.(l99$) 158:1923-1928).
  • Influenza virus is an orthomyxovirus containing eight single stranded RNA segments.
  • the eight segments code for the following proteins: HA (hemagglutinin), NA (neuraminidase), Ml (matrix), M2 (transmembrane), NP (nucleoprotein), PB2 (polymerase), PBl (polymerase), PA (polymerase), NEP (viral assembly), and NSl (interferon antagonist) (Harper et al., Clin. Med. Lab. (2002) 22:863-882; Hilleman, Vaccine (2002) 20:3068-3087; Cox et al., Scandanavian J. oflmmun. (2003) 59:1-15).
  • the most abundant protein on the virus surface is HA protein.
  • the HA protein is responsible for attachment of the virus to the sialic acid-containing receptors on the host cell surface and fusion of the viral and endosome membranes for release of the viral ribonucleotide NP (RNPs) complexes into the cytoplasm of the host cell (Cox et al., Scandanavian J. oflmmun. (2003) 59:1-15).
  • RNPs viral ribonucleotide NP
  • NA is also on the surface but in lower copy number than HA.
  • NA protein cleaves sialic acid and plays an important role in viral entry and release.
  • the M2 protein is also present on the surface (24 amino acids of the 97 amino acid protein) of the virus but is in much less abundance than HA or NA.
  • influenza virus There are three types of influenza virus, A, B and C (types are based on the sequence of NP and Ml proteins). Influenza type C causes a mild respiratory illness and is not included in current flu vaccine formulations.
  • Type B virus circulates widely among humans and is included in the current flu formulations produced each year.
  • Type B viruses have no subtypes as they contain only one type of HA and NA proteins.
  • type A viruses contain various types of HA and NA proteins that vary in sequence and, as a result, type A viruses are designated as subtypes based on the make up of these two proteins.
  • the 9 NA subtypes are infectious in humans; Hl, H2, H3, H5, H9 and Nl, N2, respectively (Cox et al., Scandanavian J. oflmmun. (2003) 59:1-15).
  • HA hemagglutinin
  • the hemagglutinin (HA) protein of the influenza virus is the most abundant protein on the surface of the virus and is primarily responsible for the humoral immune response against the virus upon infection. Therefore, HA is the leading candidate for inclusion in a subunit vaccine for influenza. While the antibody responses directed against the surface protein, HA, is a key component in a protective immune response, cellular immune responses directed against various structural and nonstructural proteins of the influenza virus are thought to also contribute to protection.
  • influenza HA protein is the primary protein found on the surface of the virus.
  • the HA found on the surface of the viron is in a trimeric form.
  • the trimer is anchored to the viral membrane by transmembrane spanning sequences at the carboxy-terminal end of each of the three monomers.
  • the main protective efficacy of influenza vaccine is attributed to anti-hemagglutinin antibodies stimulated by HA protein; the anti-HA antibodies inhibit the attachment of the virus to cells (Virelizier JL, J. Immunol. (1975) 115:434-439). Inhibition of virus attachment protects individuals against infection or serious illness depending on the magnitude of anti-hemagglutinin titers stimulated by vaccination.
  • the fusion of influenza virus to the host cell depends on the structure of the HA molecule.
  • the HA protein is c eaved ' "Mm ' ed ⁇ to the fus on peptide. This c eavage of K/v ⁇ to J ⁇ I ana jtiA2 is essential for fusion to occur (Steinhauer DA, Virology (1999) 258:1-20).
  • Another necessary step in the fusion process requires that HA trimerizes (Danieli et al., J. Cell Biol. (1996) 133:559-569). Therefore, inhibition of this viral process is very dependent on proper conformation epitopes of the HA molecule and trimers thereof, and binding of paratopes to those epitopes. This highlights the importance of raising an immune response to conformationally relevant HA protein.
  • HA is the primary protein in existing influenza vaccine formulations and influenza vaccines under development
  • the use of this protein in vaccines is confounded by the nature of HA in type A influenza viruses which are of the greatest concern.
  • Type A viruses undergo "antigenic drift” over time as the sequence in HA under goes small changes, resulting in the need to substitute "newer” strains of influenza virus in the vaccine each year to keep up with the changes in the current circulating strains (the U.S. Food and Drug Administration (“FDA”) recommends strains each year to be included in influenza vaccine for administration in the U.S.).
  • FDA U.S. Food and Drug Administration
  • antigenic shift More substantial changes in the make up of type A viruses that result from recombinations of circulating strains are referred to as "antigenic shift". These shifts are primarily in the HA gene and result in new strains being formed. As there is no pre-existing immunity to these new strains, they are often associated with pandemics of influenza (Nicholson et al., Lancet (2003) 362:1733-1745). The existence of both antigenic shift and drift pose significant challenges in preparing influenza vaccines with existing vaccine technology and for any new technology designed to produce improved influenza vaccines.
  • Influenza vaccines marketed in the United States are currently produced in embryonated chicken eggs.
  • the inactivated vaccines contain primarily hemagglutinin ("HA") protein after inactivation of live virus and purification of viral protein.
  • HA binds to a sialic acid residue on the cell to be infected.
  • the name of HA derives from the protein's ability to adhere to red blood cells and cause them to agglutinate, or clump together.
  • Inactivation of the virus is accomplished through the use of agents such as formalin, which is a compound that is known to cross-link protein and damage epitopes.
  • Influenza production procedures use of embryonated chicken eggs
  • impurities in the inactivates vaccines and preservatives added to the vaccines can lead to adverse events in those immunized with these vaccines.
  • influenza vaccine formulations are well tolerated in human subjects; mild soreness at the site of injection is the most common complaint (Margolis et al., JAMA (1990) 264:1139-1141; Nichol e ⁇ ah, Arch. Intern. Med. (1996) 156:1546-1550).
  • Manufacturers of inactivated influenza vaccines do warn individuals with allergies to eggs to avoid vaccination with the product, however, immediate hypersensitivity reactions seem to be low (James et al., J. Pedi ⁇ tr. (1998) 133:624-628).
  • Inactivated influenza vaccines have very rarely been associated with severe undesired side effects. Guillain-Barr ⁇ syndrome has been associated with influenza vaccination at a rate of one per million vaccinees (Lasky et al., N. Engl. J. Med. (1998) 339:1797-1802).
  • Inactivated influenza vaccines are 60 to 100% effective in preventing morbidity and mortality, however, lower rates of efficacy are observed in the young and elderly. In addition, reduced efficacy in the general public occurs in years of poor antigenic match of the vaccine strain to the circulating strain (Beyer et al., Vaccine (2002)20:1340-1353).
  • Suppression or impairment of either the humoral or cell mediated branch of the immune system can lead to increased susceptibility or severity of disease induced by infectious agents (e.g., opportunistic infections).
  • infectious agents e.g., opportunistic infections.
  • Ih "immunosuppressed” individuals the immune response is prevented or diminished (e.g., by administration of radiation, antimetabolites, antilymphocyte serum, or specific antibody).
  • "Immunocompromised” or “immunodeficient” individuals have their immune system attenuated (e.g., by malnutrition, irradiation, cytotoxic chemotherapy, or diseases such as cancer or AIDS, or by primary immune deficiencies).
  • Immunosuppressed, immunocompromised, immunosenescent, and non-suckling infant populations are at particular risk for many infectious diseases, but concomitantly are too vulnerable to the effects i r u a a enua e ve v rus vac es, an e are an important targe audience for vaccine development.
  • infectious diseases e.g., influenza infection - Katz et al., supra
  • immunodeficient population are at particular risk for many infectious diseases, but concomitantly are too vulnerable to the effects i r u a a enua e ve v rus vac es, an e are an important targe audience for vaccine development.
  • the fact that members of the immunodeficient population have some degree of immune impairment makes the challenge of developing an immunogenic and protective vaccine for the immunodeficient population particularly difficult.
  • influenza vaccine inherently limits the amount of vaccine that can be made in time for the upcoming flu season.
  • the two major suppliers of flu vaccine for the United States are Aventis (Fluzone®) and Chiron (Fluvirin®). Both companies produce influenza virus in embryonated chicken eggs (90 million of them used per year for manufacture).
  • the virus is harvested, inactivated (formaldehyde, and betapropiolactone, respectively), filtered, and purified by continuous zonal centrifugation.
  • the resultant product is standardized by the HA content and contains 15 ⁇ g of each HA antigen subtype.
  • Various other flu proteins are also contained in the vaccine in lower and various amounts. Inactivation steps tend to damage antigen epitopes, which in turn requires the use of more protein to provide an adequate immune response.
  • the current inactivated vaccine formulations are not adjuvanted.
  • inactivated-virus vaccines for pandemic influenza strains is further complicated by the need to grow the virus strains under BSL-3 level conditions.
  • avian strains of influenza are lethal to chicken embryos, necessitating the construction of suitable strains using reverse genetics that can be used for manufacture in embryonated chicken eggs (Wood, Vaccine (2002) 20:B40-B44).
  • influenza vaccines protective immunity is considered to be achieved if an individual mounts an anti-hemagglutinin titer of >1 :40 and seroconversion to the influenza immunizing strain is considered to occur if a four-fold increase in titer is achieved.
  • the level of anti-NA antibodies necessary to limit viral spread has not yet been defined (Ada and Jones, Curr. Topics Microbiol Immunol. (1986) 128:1-54; Aymard-Henry et. al., Bull WHO (1973) 48:199-202; Beran et. al., Centr. Eur. J. Pub.
  • BES Baculovirus expression system
  • insect cells are infected with baculovirus carrying the gene to .
  • purification as insect cell proteins are co-purified with the expressed protein and cellular enzymes are released that can degrade the desired protein products.
  • Medlmmune's FluMist® is a newly licensed live attenuated vaccine that is administered by nasal spray to patients between the ages of 5 and 49. This new vaccine is not licensed for use in "at-risk" populations. Medlmmune produced approximately 4 million doses of FluMist® vaccine for the 2003 flu season. This vaccine is also grown on embryonated chicken eggs. This vaccine is a live attenuated formulation that is delivered by nasal spray. Besides limitations in the amount of doses that can be manufactured each year, the vaccine is not licensed for use in the young and elderly populations, which need protection from influenza the most.
  • Antiviral compounds are available for combating influenza infections; however, they come with limitations on their use (Williams et al., KaohsiungJ. Med. Sd (2002) 18:421-434). Amantadine and rimantadine are effective for the prevention and treatment of influenza infection; however, they are only effective for type A viruses. Drug resistant virus strains have also been isolated from individuals treated with these compounds (Englund et al., Clin. Infec. Dis. (1998) 26: 1418-1424). These drugs also have undesirable side effects (Dolin et al., N. Engl. J. Med. (1982) 307:580-584).
  • Newer antiviral agents such as zanamivir (nasal spray) and oseltamivir (oral) block (by transition-state analog inhibition) influenza A and B enzyme NA. These drugs can prevent disease if given prophylactically and can lessen the duration of symptoms if given within 48 hours of infection. Zanamivir and oseltamivir have fewer side effects but are more expensive than amantadine and rimantadine. Oseltamivir (trade name, Tamiflu®) is marketed by Roche Holding AG, who is building a new production plant devoted to production of oseltamivir.
  • influenza vaccine clearly are limited in meeting the increasing demand for a higher number of doses per year and for addressing needed improvements in the immunogenicity and efficacy in certain segments of the population.
  • DNA vaccines encoding the HA and NP genes have been evaluated in mouse challenge models (Williams et al., KaohsiungJ. Med. ScL (2002) 18:421-434; Kemble and Greenberg, Vaccine (2003) 21:1789-1795).
  • Vaccination with DNA encoding the NP gene resulted in protection from challenge with a heterologous influenza strain (Montgomery et al., DNA Cell Biol. (1993) 12:777-783). Protection from homologous virus challenge was accomplished after vaccination with DNA encoding HA in mice.
  • DNA vaccines encoding the influenza HA, M2, and NP genes have been evaluated as alternative vaccines for influenza. This method is obviously not dependent on eggs or mammalian cell culture. Most studies have only presented encouraging results in mice (Montgomery et al., 1993; Ulmer et al., Science (1993) 259:1745-1749; and Williams et al., KaohsiungJ. Med. ScU (2002) 18:421-434). Reports of promising results in larger animals are very hard to find. As an example a M2-NP DNA that worked well in mice appears to have exacerbated disease following challenge in a pig model (Heinen et al., J. Gen. Virol.
  • HA the primary component for influenza vaccines has proven to be a difficult protein to express as a recombinant.
  • Expression in Pichia of a membrane anchorless HA molecule has been reported (Saelens et al., Eur. J. Biochem. (1999) 260(1): 166-175). While the expressed HA protein had appropriate structure based on antibody binding and resulted in partial protection when used to immunize mice, the product was not completely uniform in nature. The N-terminus was variable due to variable processing and the glycosylation patterns where heterogeneous also. Despite statements that the Pichia expressed HA protein has potential as a vaccine candidate there is no indication that this effort has been carried on for testing in humans.
  • BES baculovirus expression system
  • An early report on the expression of full length HA using BES resulted in HA being localized on the surface of the insect cells (Kuroda et al., EMBO J. (1986) 6:1359-1365). Further studies were reported on the expression of soluble HA from BES (Valandschoot et al., Arch Virol (1996) 141:1715-1726). This report on soluble baculovirus expressed HA like the Pichia expressed HA determined that the protein had some native-like characteristics, but was mostly aggregated and did not provide any protection when tested in a mouse model.
  • the recombinant baculovirus-expressed HA proteins under development by Protein Sciences Corporation represent the most advanced recombinant influenza vaccines to date.
  • the HA expressed by PSC represents the full length molecule and results in the localization on the host insect cells.
  • the HA is purified through a series of steps following extraction from the membrane.
  • An H5 HA vaccine based on this methodology has been evaluated in human clinical trials (Treanor et al., Vaccine (2001)19:1732-1737).
  • One hundred forty seven healthy adults were randomly assigned to receive two intramuscular injections of either 25, 45 or 90 ⁇ g each, one dose of 90 ⁇ g followed by a dose of 10 ⁇ g, or two doses of placebo; doses given at intervals of 21, 28 or 42 days.
  • the vaccine was not adjuvanted.
  • the clinical trial demonstrated that a neutralizing antibody titer of > 1 :80 was achieved in some individuals receiving a single dose of 90 ⁇ g (23%) or two g . ors o is pa onc u e nogemcity oi the vaccine needs to be improved.
  • VLP virus-like particles
  • BES virus-like particles
  • This methodology is currently being pursued by Novavax (Malvern, PA).
  • VLPs consisting of HA, NA and Ml proteins have been produced and are being developed for use as vaccines (Pushko et al., Vaccine (2005) 23(50):5751-5759).
  • the VLPs exhibit functional characteristics of influenza virus and were shown to inhibit replication of influenza virus after challenge of vaccinated Balb/c mice.
  • the use of VLPs for influenza vaccination appears promising; however, the authors do cite manufacturing issues that need to be solved in order to develop a scalable manufacturing process that could be used to meet production needs.
  • a recombinant expression system be able to produce both a high quality product and high yields of the desired product.
  • the Drosophila expression system as defined below, was selected by the inventors for the expression of influenza recombinant subunit proteins. This system has been shown to be able to express heterologous proteins that maintain native-like biological structure and function (Bin et al, Biochem J. (1996) 313:57-64 and Incardona and Rosenberry, MoI. Biol, of the Cell (1996) 7:595-611).
  • the Drosophila expression system is also capable of producing high yields of product.
  • the use of an efficient recombinant expression system will ultimately lower the cost per dose of a vaccine and enhance the commercial potential of the product.
  • using the Drosophila expression system to produce influenza HA and Ml proteins is novel.
  • the invention provides recombinant influenza subunit proteins and immunogenic compositions that can be utilized as vaccines to afford protection against influenza in animal models and humans.
  • the recombinant subunit proteins of the invention are expressed from stably transformed insect cells that contain integrated copies of the appropriate expression cassettes in their genome.
  • the insect cell expression system provides high yields of recombinant subunit proteins with native-like conformation.
  • the recombinant subunit proteins of the invention represent full length or truncated forms of the native influenza proteins. Additionally, multimeric forms of several of the recombinant subunit proteins have been produced. Specifically, the subunits are derived from the HA and Ml proteins of influenza.
  • subunit proteins are secreted from the transformed insect cells and then purified from the culture medium following the removal of the host cells. Avoiding lysis of the host cells by either viral means or by physical means simplifies purification, improves yields, and avoids potential degradation of the target protein.
  • the invention also provides for the use of adjuvants as components in an immunogenic composition compatible with the purified proteins to boost the immune response resulting from vaccination.
  • adjuvants are selected from the group comprising saponins (e.g, GP-OlOO), or derivatives thereof, emulsions alone or in combination with carbohydrates or saponins, and aluminum-based adjuvants (collectively, "alum” or “alum-based adjuvants”) such as aluminum hydroxide, aluminum phosphate, or a mixture thereof.
  • Aluminum hydroxide commercially available as "Alhydrogel” was used as alum in the Examples.
  • a ⁇ iun is any pian g ion a ca o ie a sugar an a sapogenin aglycone.
  • Sapogenin is the nonsugar portion of a saponin. It is usually obtained by hydrolysis, and it has either a complex terpenoid or a steroid structure that forms a practicable starting point in the synthesis of steroid hormones.
  • the saponins of the invention can be any saponin as described above or saponin-like derivative with hydrophobic regions, especially the strongly polar saponins, primarily the polar triterpensaponins such as the polar acidic bisdesmosides, e.g.
  • Nutanoside, Dianthoside C, Saponaside D, aescine from Aesculus hippocastanum or sapoalbin from Gyposophilla struthium preferably, saponin extract Quillaja saponaria Molina and Quil A.
  • saponin may include glycosylated triterpenoid saponins derived from Quillaja Saponaria Molina of Beta Amytin type with 8-11 carbohydrate moieties as described in U.S. Patent No. 5,679,354.
  • Saponins as defined herein include saponins that may be combined with other materials, such as in an immune stimulating complex ("ISCOM")-like structure as described in U.S. Patent No. 5,679,354.
  • ISCOM immune stimulating complex
  • Saponins also include saponin-like molecules derived from any of the above structures, such as GPI-0100, such as described in U.S. Patent No. 6,262,029.
  • the saponins of the invention are amphiphilic natural products derived from the bark of the tree, Quillaia saponaria.
  • they consist of mixtures of triterpene glycosides with an average molecular weight (Mw) of 2000.
  • Mw average molecular weight
  • a particularly preferred embodiment of the invention is a purified fraction of this mixture.
  • the invention further provides methods for utilizing the vaccines to elicit the production of antibodies against the various types and subtypes of influenza virus in a mammalian host as a means of conferring protection against influenza.
  • the vaccine formulations are shown to induce strong overall antibody titers, as well as strong hemagglutinin-inhibition antibody titers, in comparison to other formulations.
  • the vaccine formulations are shown to provide protection against influenza challenge in a mouse model.
  • the proteins produced by the invention have increased immunogenicity and efficacy, are less costly to produce, and have a shorter production cycle.
  • FIG. 1 Lymphocyte proliferation of antigen stimulated splenocytes
  • FIG.2. IFN- ⁇ production from antigen stimulated splenocytes.
  • FIG. 4. H5 HA ELISA antibody titers.
  • FIG. 5. H3 HA ELISA antibody titers.
  • the invention provides influenza recombinant subunit proteins that are produced and secreted from stable insect cell lines that have been transformed with the appropriate expression plasmid.
  • the recombinant proteins are used individually or combined together with or without adjuvant(s) such that they are effective in inducing a strong antibody response capable of inhibiting hemagglutination in in vitro assays. This antibody response is indicative of in vivo protection against influenza infection.
  • the recombinant proteins When used in combinations, in addition to inducing relevant antibody responses, the recombinant proteins also induce cellular immune responses which further enhance the efficacy of the vaccine formulation.
  • the use of appropriate antigens, with or without adjuvants or adjuvant combinations can be used to induce a specific immune response that results in antibodies that are capable of providing protection from influenza.
  • the recombinant influenza subunit proteins that are a component of the vaccine formulation described herein are produced in a eukaryotic expression system that utilizes insect cells.
  • Insect cells are an alternative eukaryotic expression system that provides the ability to express properly folded and post-translationally modified proteins while providing simple and relatively inexpensive growth conditions.
  • the majority of insect cell expression systems are based on the use of baculovirus-derived vectors to drive expression of recombinant proteins.
  • Expression systems using baculovirus-derived vectors are not based on the use of stable expression cell lines. Instead these systems rely on the infection of host cells for each production cycle.
  • BiotechnoL (1991) 2:704-707; Gulp, J.S., et al., Biotechnology (NY) (1991) 9:173-177) is an insect cell expression system based on the generation of stably transformed cell lines for recombinant protein expression.
  • This insect cell expression system has been shown to successfully produce a number of proteins from different been shown to maintain structural and functional characteristics of the corresponding native proteins.
  • proteins that have been successfully expressed in the Drosophila expression system include HIV g ⁇ l20 (CuIp, J.S., et aL, Biotechnology (NY) (1991) 9:173- 177; Ivey-Hoyle, M., Curr. Opin.
  • HBI has also determined that subunit proteins produced from the Drosophila expression system produced superior immunogenic material.
  • a comparison of Plaque Reduction Neutralization Titers (PRNTso) between comparable -Dr ⁇ s ⁇ p/u/ ⁇ -expressed dengue E protein and Pi'c ⁇ i ⁇ -expressed dengue E protein showed ranges of 1 :400 - 1:1600 and ⁇ l:10 - 1:80, respectively for the two systems, using equivalent doses for immunization.
  • PRNTso Plaque Reduction Neutralization Titers
  • the insect cells used as host cells for expression of the influenza recombinant subunit proteins are or are derived from the Drosophila melanogaster S2 cell line (Schneider, J. Embryol. Exp. Morph. (1972) 27:353-365).
  • the Drosophila expression system provides a stable and continuous insect cell culture system that has the potential to produce large quantities of native-like subunit proteins that maintain relevant immunological properties.
  • the focus of the present invention is on two specific influenza type A subtypes, H3N2 and H5N1.
  • H3N2 subtype the A/Fujian/411/02 influenza strain was used as the source for HA gene.
  • H5N1 subtype two strains were used, A/Hong Kong/156/97 and A/Indonesia/5/05.
  • the A/Hong Kong/156/97 strain was used as the source for HA and Ml while the A/Ihdonesia/5/05 was used for only HA sequences.
  • nucleotide sequences encoding the various proteins of these specific influenza strains as well as most other strains are available in the GenBank (www.ncbi.nlm.nih.gov) and ISD (www.flu.lanl.gov) databases.
  • GenBank www.ncbi.nlm.nih.gov
  • ISD www.flu.lanl.gov
  • the expression and secretion of the influenza subunit proteins HA and Ml from Drosophila S2 cells was evaluated by operably linking the coding sequences of such proteins to a secretion signal sequence such that the expressed products were secreted into the culture medium.
  • the tPA tissue plasminogen activator
  • All nucleotide sequences encoding the described influenza subunit proteins were made synthetically (DNA2.0, Menlo Park, CA) and were derived from sequences available in the GenBank and ISD databases. The specifc synthetic DNA sequences encoding the influenza subunit proteins were also codon optimized for expression in insect cells.
  • the subunit protein encoding sequences described herein were cloned into Drosophila expression plasmids under the control of the Drosophila MtnA (metallothionein) promoter utilizing standard recombinant DNA methods.
  • the Drosophila expression plasmids containing the cloned influenza sequences were then used to transform Drosophila S2 cells.
  • the HA protein was truncated at the C-terminal end to remove the membrane spanning region to allow for secretion of a soluble subunit.
  • the soluble membrane anchor-less subunit is referred to as the HA ectodomain (surface exposed region of a transmembrane anchored protein).
  • the truncated and secreted HA subunits are designed to maintain native-like characteristics of the exposed portion of the membrane anchored HA as displayed on the surface of the virus and are capable of eliciting a strong immune response . and approximately two thirds of the HA2 region (truncation is in the HA2 region).
  • the H3 HA protein was truncated at amino acid G-V 5 2 0 and the H5 HA protein was truncated at amino acid Grjt ⁇ i of the full-length sequences (includes the secretion signal).
  • the C-terminal portion so truncated at amino acid GIV 5 2 0 in the case of H3 HA protein, and at Gly5 2 i in the case of H5 HA protein, is called herein a "nominal ectodomain".
  • the truncation point can be varied up to 10% of the length of a nominal ectodomain so long as such variation does not affect conformation of the epitopes of the remaining soluble HA subunit protein (ectodomain).
  • the native secretion signal sequences were removed for expression as a heterologous secretion signal (tPA) provided by the expression plasmid was utilized to direct secretion of the influenza subunits.
  • the H3 HA ectodomain protein sequence expressed is SEQ DD NO: 1 and the H5 HA Hong Kong and Indonesia ectodomain protein sequences expressed are SEQ ID NO:2 and SEQ ID NO:3, respectively.
  • the HA ectodomain subunits are referred to by the HA subtype from which they are derived followed by HA-Ecto, for example H3 HA-Ecto.
  • HA subunits consisting of further truncations of the HA molecule, i.e., truncations that remove a larger amount of the C-terminal end beyond that removed by the ectodomain and segments of the N-terminal end of the HA sequence is described below.
  • These further truncation of HA are designed to express HA subunits that result in a more focused immune response to the naturally exposed surfaces of the HA molecule upon immunization.
  • Such further truncations of the ectodomain are produced by removing the entire HA2 region (the C-terminal region representing approximately one-third of Ml length HA protein) and a small segments of the N-terminal region of HA.
  • the N- and C-terminally truncated subunits encompass the HA region known as the globular heads and are therefore referred to as HA-heads.
  • the C-terminal truncation is at constant point for all "head” subunits. Specifically the "head” subunits are truncated at Arg 329 for H3 HA-heads and Arg 3 2 ⁇ for H5 HA-heads (the number of amino acids for this purpose is based on the mature HA protein and does not include the secretion signal).
  • the specified N- and C-terminal truncations for both the H3 and HS HA-heads are called here in "nominal HA-heads".
  • Both the N- and C- terminal truncation points can be varied up to 10% of the length of the nominal HA-heads so long as such variation does not affect the conformation of the epitopes on the remaining soluble HA-head.
  • the "head" subunits are distinguished by the position of the N-terminal truncation. For example a subunit named ' ⁇ 3 HA-Al 9-head" is one derived from the H3 subtype and is N-terminally truncated at AIaI 9 (A19). Again, the numbering is based on the mature HA protein.
  • the HA-head sequences expressed are shown in Alignments 1 and 2 of Appendix A for H3 and H5 respectively, relative to the corresponding HA octodomain sequence.
  • the amino acid sequence of H3 HA-Al 9-head is SEQ ID NO:4.
  • the amino acid sequence of H3 HA-G49-head is SEQ ID NO:5.
  • the amino acid sequence of H5 HA-A9-head is SEQ ID NO:6.
  • the amino acid sequence of H5 HA-G39-head is SEQ ID NO:7.
  • a multimeric form of HA was expressed.
  • HA sequences analogous to the HA ectodomains described above were further modified by fusing an amino acid sequence of 36 residues to the C-terminal end of the HA ectodomain sequence.
  • the fused, and secreted HA s ⁇ bunits that form trimeric molecules are shown to maintain native-like characteristics of the HA protein as it is displayed on the surface of the virus and are capable of eliciting a strong immune response when combined in a vaccine formulation.
  • the foldon sequence which is located at the C-terminus of the fibritin protein, naturally brings together three monomers of fibritin via non-covalent bonding to form a trimeric molecule.
  • the "HA foldons” were constructed by fusing the C-terminal end of the ectodomain (GIV 52 0 for H3 and GIV 521 for H5) to the 36 amino acid foldon containing sequence.
  • H3 HA-foldon and H5 HA-foldon are collectively known as "HA-foldons" and individually as an ' ⁇ A- foldon".
  • the protein sequence expressed for the H3- and H5-foldon subunits are shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively.
  • the H5N1 Ml subunit representing the full length native Ml protein was expressed.
  • the Ml protein is encoded by amino acids 1 to 252.
  • the Ml protein sequence expressed is shown in SEQID NO:10.
  • the amino acid sequences of SEQ ID NOS: 1 to 10 can have up to 10% substitution in residues so long as such substitutions do not affect conformation of the epitopes.
  • influenza recombinant subunit proteins that are expressed and secreted from the stably transformed S2 cell lines, as described below and utilized in the preferred vaccine formulations, are first purified by a variety of methods, as described below.
  • the preferred purification method produces protein that maintains its native conformation.
  • a vaccine formulation that combines the Drosophila- expressed influenza recombinant subunit proteins as described herein, with or without one or more adjuvants, potentiates a strong immune response.
  • the use of such a vaccine formulation induces strong hemagglutinin antibody titers, e.g., > 1:40.
  • the unique ability of such a vaccine formulation to elicit high hemagglutinin antibody titers is supported by the fact that other recombinantly expressed influenza proteins failed to induce potent immune responses.
  • the vaccine formulation is capable of conferring protection from influenza challenge in the mouse model. Further details that describe the characteristics of the individual components and the remarkable efficacy of this vaccine formulation are contained below.
  • the vaccine formulation is characterized by the use of low doses of recombinant subunit proteins capable of eliciting a specific and potent immune response. Low doses are defined as 15 ⁇ g or less of recombinant protein. This is in contrast to other influenza recombinant subunit proteins that have required higher doses to achieve moderate immune responses.
  • the present invention thus concerns and provides a vaccine formulation as a means for preventing or attenuating infection by influenza viruses.
  • a vaccine is said to prevent or attenuate disease if its administration to an individual results either in the total or partial immunity of the individual to the disease, i.e., a total or partial suppression of disease symptoms.
  • a vaccine formulation containing one or more subunits is administered to a subject by means of conventional immunization protocols involving, usually but not restricted to, multiple administrations of the vaccine.
  • the use of the immunogenic compositions of the invention in multiple administrations may result in the increase of antibody levels and in the diversity of the immunoglobulin repertoire expressed by the immunized subject.
  • Administration of the immunogenic composition is typically by injection, e.g., intramuscular or subcutaneous; however, other systemic modes of administration may also be employed.
  • an "effective dose" of the immunogenic composition is one that is sufficient to achieve a desired biological effect.
  • the dosage needed to provide an effective amount of the composition will vary depending upon such factors as the subject's age, genetic background, condition, and sex.
  • the immunogenic preparations of the invention can be administered by either single or multiple dosages of an effective amount. Effective amounts of the compositions of the invention can vary from 1-100 ⁇ g per dose, more preferably from 1-15 ⁇ g per dose.
  • influenza subunits HA and Ml are primarily directed at the expression of the influenza subunits HA and Ml from the type A - A subtypes and to influenza types B and C.
  • influenza subunit proteins HA and Ml proteins utilizing stably transformed insect cell lines.
  • Drosophila expression system is utilized.
  • the purification of the expressed recombinant subunit proteins is also demonstrated.
  • the Examples demonstrate that the Drosophila expressed recombinant proteins when used as immunogens result in robust and biologically relevant immune responses.
  • the results presented demonstrate that individual influenza subunit proteins derived from the native influenza proteins HA and Ml or various combinations of these same subunit proteins are capable of providing enhanced protection from challenge in mouse models.
  • the utilization of recombinantly expressed HA and Ml proteins from stably transformed insect cells results in superior immunogenic compositions and meets the need and solves the technical problem set forth above.
  • the pMttbns expression vector contains the following elements: the Drosophila metallothionein promoter (Mm), the human tissue plasminogen activator (tPA) signal sequence, and the SV40 early polyadenylation signal (Gulp et al, Biotechnology (1991) 9:173-177).
  • the pCoHygro plasmid provides a selectable marker for hygromycin (Van der Straten, Methods in MoI. and Cell Biol. (1989) 1:1-8).
  • the hygromycin gene is under the transcriptional control of the Drosophila COPIA transposable element long terminal repeat.
  • the pMttbns vector was modified by deleting a 15 base pair BamHI fragment which contained an extraneous Xho I site.
  • This modified vector referred to as pMtt ⁇ Xho, allows for directional cloning of inserts utilizing unique BgI ⁇ and Xho I sites.
  • the Drosophila expression system has been reported to express high levels of properly folded proteins (CuIp et al Biotechnology (1991) 9:173-177, Bernard et al Cytotechnology (1994)15:139-144, Bin et al Biochem J. (1996) 313:57-64, Incardona and Rosenberry, MoI. Biol of the Cell (1996) 7:595-611).
  • Expression vectors based on the Drosophila metallothionein (Mtn) promoter provide regulated expression of heterologous proteins (Van der Straten, Methods in MoI. and Cell Biol (1989) 1:1-8), Johansen, H. et al., Genes Dev.
  • the Drosophila expression plasmids encoding the influenza subunit proteins were constructed by inserting defined segments of the appropriate genes in the Drosophila expression vector pMtt ⁇ Xho.
  • the appropriate regions of the influenza genes were generated by gene synthesis (DNA2.0, Menlo Park, CA). In addition to the synthesis of appropriate genes of interest, the genes were also codon optimized for expression in insect cells.
  • the synthetic genes also included appropriate restriction endonuclease cleavage sites for cloning along with necessary control elements, such as stop codons.
  • the synthetic influenza genes were cloned into the pMtt ⁇ Xho vector digested with BgIII and Xhol.
  • Drosophila S2 cells (Schneider, J. Embryol Exp. Morph. (1972) 27:353-365) obtained from ATCC were utilized in the S2 system. Cells were adapted to growth in Excell , , were in Excell 420 medium. Cells were passed between days 5 and 7 and were typically seeded with expression plasmids at a density of 1x10 6 cells/ml and incubated at 26 0 C. Expression plasmids containing sequences encoding influenza subunit proteins were transformed into S2 cells by means of the calcium phosphate method.
  • the cells were co- transformed with the pCoHygro plasmids for selection with hygromycin B at a ratio of 20 ⁇ g of expression plasmid to 1 ⁇ g of pCoHygro. Following transformation, cells resistant to hygromycin, 0.3 mg/ml, were selected. Once stable cell lines were selected, they were evaluated for expression of the appropriate products. Five ml aliquots of culture medium were seeded at 2x10 6 selected cells/ml, induced with 0.2 mM CuSO 4 , and cultured at 26°C for 7 days. Cultures were evaluated for expression of subunit proteins in both the cell associated fractions and the culture medium.
  • Proteins were separated by SDS-PAGE and either stained with Coomassie blue or blotted to nitrocellulose. Antibodies specific for a given target protein being expressed were used to probe Western blots. Expression levels of 1 mg/L or greater are readily detected in Drosqphila cultures by Coomassie staining of SDS-PAGE gels. To produce larger volumes of product, the transformed Drosophila S2 cells were grown as suspension cultures in spinner flasks or bioreactors.
  • the full length HA gene (HAO) of the H3N2 strain A/Fujian/411/02 encodes a protein of 566 amino acid residues.
  • the sequence utilized was derived from the nucleotide sequence in accession number ISDN38157 (ISD, www.flu.lanl.gov)>
  • the non- truncated protein sequence contains a 16 amino acid secretion signal sequence at the N- terminus and a C-terminal membrane anchor.
  • H3 HA-Ecto soluble H3 HA ectodomain
  • the pMtt ⁇ Xho expression plasmid containing ("loaded with") the synthetic gene for the H3 HA-Ecto subunit protein was used to transform S2 cells. Upon selection of stable cell lines the cells were screened for expression of the secreted form of the H3 HA-Ecto protein. The expression of the described H3 HA-Ecto subunit resulted in a uniform product of the expected molecular weight. The glycosylation pattern of the secreted H3 HA-Ecto is uniform as the treatment with PNGase results in a shift that is consistent with the presence of 7 glycosylation sites.
  • the expression level of the H3 HA-Ecto target protein secreted into the culture medium of S2 cells has been estimated to be between 30 and 40 ⁇ g/ml.
  • j i protein of 568 amino acid residues.
  • the sequences utilized are derived from the nucleotide sequence in accession number AF046088 (Genbank, www.ncbi.nlm.nih.gov).
  • the HAO protein sequence contains a 16 amino acid secretion signal sequence at the N-terminus and a C-terminal membrane anchor.
  • a soluble H5 HA molecule (ectodomain) an N- and C- truncated molecule was expressed that is contained the sequence from Asp 17 to GIV 521 (residue 175 of HA2, analogous to the C-terminus of the X31 crystal structure, Wilson et al. Nature (1981) 289:366-367), of the full length protein.
  • the HA of the A/Hong Kong/156/97 (H5N1) strain contains a stretch of 6 basic amino acid residues at the HA1/HA2 junction that encodes a furin cleavage site. This site is cleaved upon expression in S2 cells.
  • the pMtt ⁇ Xho expression plasmid containing ("loaded with") the synthetic gene for the H5 HA-Ecto subunit protein was used to transform S2 cells. Upon selection of stable cell lines the cells were screened for expression of the secreted form of the H5 HA-Ecto protein.
  • the expression of the described H5 HA-Ecto subunit resulted in a product consisting of a number of bands (+ or - 10 kD) in the range of the expected molecular weight under non- reducing conditions.
  • the glycosylation pattern of the secreted H5 HA-Ecto appeared to be uniform based on the treatment with PNGase which results in a shift that is consistent with the presence of 5 glycosylation sites under reducing condtions. Therefore, the multiband pattern of expression appears to be the result of variations in folding of the molecule.
  • the expression level of the H5 HA-Ecto target protein secreted into the culture medium of S2 cells has been estimated to be approximately 5 ⁇ g/ml.
  • the HA protein for both H5N1 strains contains a stretch of basic amino acid residues at the HA1/HA2 junction that encodes a furin cleavage site. This site is cleaved upon expression in S2 cells.
  • Alternative forms of the H5 HA-Ecto were also expressed. These alternative forms were made by creating a mutation within the furin cleavage site which prevented the protease cleavage of the H5 HA-Ecto subunits upon expression.
  • the pMtt ⁇ Xho expression plasmid containing the synthetic gene for the H5 HA- Ecto-mut subunits were used to transform S2 cells. Upon selection of stable cell lines the cells - - - m. ne expression of the H5 HA-Ecto-mut subunits resulted in a more uniform protect than that of the H5 HA-Ecto subunits. .
  • the expression level of the H5-HK-HA Ecto and H5-Indo-HA Ecto proteins secreted into the culture medium of the S2 cultures has been estimated to be 5 to 10 ⁇ g/ml and 10 to 15 ⁇ g/ml, respectively.
  • H5 HA-Ecto, H3 HA-Ecto, and derivatives thereof are collectively known as "hemagglutinin ectodomain protein subunits" and individually as an “hemagglutinin ectodomain protein subunit”.
  • Non-immunoaffinity purification approaches such as the method of Vanlandschoot et al. (Arch. Virol. (1996) 141:1715-1726), which was originally used to purify A/Victoria/3/75 (H3N2) HA expressed as a secreted product in Spodopterafrugiperda-9 (Sf9) cells, were also evaluated for the purification of secreted influenza HA-Ecto subunit from the S2 culture supernatant. For H3 HA-Ecto subunit a two step purification method was developed.
  • the bulk harvest was diluted 1/3 with buffer A (2OmM sodium phosphate, pH 7.0) then loaded onto a SP-sepharose (GE Healthcare, Piscataway, NJ) column, which was subsequently washed with wash buffer B (5OmM sodium phosphate, pH 7.0) until baseline absorbance was achieved.
  • Bound H3 HA-Ecto was eluted with buffer B containing 0.5M NaCl.
  • the elution product from the SP-sepharose was then diluted 1/2 with buffer C (0.1M sodium phosphate, pH 7.0) then loaded onto a ceramic hydroxyapatite column (CHT; Bio-Rad Laboratories, Hercules, CA), which was then washed with buffer C until baseline absorbance was achieved.
  • Bound H3 HA ectodomain was eluted with 0.5M sodium phosphate, pH 7.0.
  • the product was concentrated and buffer exchanged by ultrafiltration for characterization.
  • the H5 HA-Ecto and H5 HA-Ecto-mut subunits were purified by a three step chromatographic process.
  • the bulk harvest was diluted 1/4 with buffer A (25mM Tris-HCl, pH 8.8, + 0.05% tween-20) then loaded onto a CHT column, which was subsequently washed .
  • buffer A 25mM Tris-HCl, pH 8.8, + 0.05% tween-20
  • the elution product was loaded onto a Q-sepharose (GE Healthcare, Piscataway, NJ) column equilibrated against buffer A. The column was washed with buffer A then with buffer A containing 5OmM NaCl.
  • the bound H5 HA-Ecto was eluted with buffer A containing IM NaCl.
  • the Q-sepharose product was further fractionated by size exclusion chromatography on a Sephacryl S-100 column (1.5 x 95.5cm) using 1 ImM phosphate buffered saline (14OmM NaCl), pH 7.2, for column buffer.
  • the fractions containing H5 HA-Ecto were pooled and concentrated for characterization.
  • the ectodomain subunits described in Example 1 were further truncated at both the N- and C-terminal ends.
  • the N- and C-terminally truncated subunits encompass the HA region known as the globular heads and are therefore referred to as HA-heads.
  • the C- terminal truncation is at constant point for all "head" subunits.
  • the "head" subunits are truncated at Arg 329 for H3 HA-heads and Arg32 6 for H5 HA-heads (the number of amino acids for this purpose is based on the mature HA protein-does not include the secretion signal-as opposed to the numbering in Example 1 which is based on the full length sequence containing the secretion signal).
  • Two N-terminal truncations were made for both H3- and H5-heads. While the numbering of the truncations between the two subtypes does not match, the truncations are equivalent based on alignment of the protein sequences. The first N- terminal truncation is made at an Ala residue, Alag for H5 and AIa ⁇ for H3.
  • the second N- terminal truncation is made at a GIy residue, Gly 3 g for H5 and GIV49 for H3.
  • the "head" subunits are designated by the position of the N-terminal truncation, specifically for the above described truncations the subunits are referred to as H5 HA-A9-head, H5 HA-G39, H3 HA- A19-head, and H3-HA-G49-head.
  • the methods used to clone, transform, express and characterize the HA-head subunits are the same as those described in Example 1. Upon selection of stable cell lines, the cells were screened for expression of the secreted form of the HA-heads. The expression of the described HA-head subunits resulted in a uniform product of the expected molecular weight for H5 derived heads where as expression of H3 derived heads resulted in multiple bands in the a range (+ or - 10 kD) of the expected molecular weight. The expression level of the H3 HA- heads and H5 HA-heads secreted into the culture medium of o the S2 cultures has been approximately 5 ⁇ g/ml and 20 ⁇ g/ml, respectively.
  • H5 HA-heads Purification of H5 HA-heads was accomplished by a non-immunoaffinity purification method. Bulk harvest was diluted 1/3 in buffer A (2OmM sodium phosphate, pH 6.2) then loaded onto a CHT column, which was washed with buffer A until baseline absorbance was achieved. The unbound material in the flow-through, which contained the H5 HA-heads, was loaded directly onto a SP-sepharose column, which was washed with buffer A until baseline absorbance was achieved. Bound H5 HA-heads were eluted with buffer A containing 0.1M NaCl.
  • the elution product was then polished by size exclusion chromatography on a Sephacryl S-100 (GE Healthcare, Piscataway, NJ) column (1.5 x 95.5cm) using 1 ImM phosphate buffered saline (14OmM NaCl), pH 7.2, for column buffer.
  • the fractions containing H5 HA-heads were pooled and concentrated for characterization.
  • the HA foldons were constructed by fusing the C-terminal end of the ectodomain (Glys 2 o for H3 and Gfy&i for H5) to the 36 amino acid foldon containing sequence. The expression of this fusion of the HA ectodomain to the foldon sequence results in the production of a soluble non-covalently linked trimeric HA subunit.
  • the HA foldons are referred to by the HA subtype from which they are derived and followed by HA-foldon, for example "H5 HA foldon.”
  • HA-foldon subunits [080] The methods used to clone, transform, express and characterize the HA-foldon subunits are the same as those described in Example 1. Upon selection of stable cell lines, the cells were screened for expression of the secreted form of the HA-foldons. The expression of the described HA-foldon subunits resulted in a uniform product of the expected molecular weight. The expression level of both H3 HA-foldon and H5 HA-foldon secreted into the i respectively.
  • the H3 HA-foldon was purified using a two step chromatographic method. Bulk harvest was. diluted 1/4 with buffer A (2OmM Tris-HCl, pH 8.0) then loaded onto a Q- sepharose column that had been equilibrated against buffer A. The column was then washed with buffer B (2OmM Tris-HCl, pH 5.0) until baseline absorbance was achieved. Bound material was then eluted by washing the column with buffer B containing 0.125M NaCl and IM NaCl. The 0.125M NaCl fraction, which contained the H3 HA-foldon was diluted 1/2 with buffer B then loaded onto a SP-sepharose column equilibrated against buffer B.
  • IAC is the preferred method of purification for H5 HA foldon.
  • the current method used to purify the H5 HA foldon is based on the methods developed for the purification of H5 HA ectodomain and H3 HA foldon which utilize Q-sepharose, SP-sepharose, and CHT chromatographic matrices.
  • Ml The full length Ml gene from the H5N1 strain A/Hong Kong/156/97 encodes a protein of 252 amino acids.
  • Ml is derived from the influenza M sequence that also encodes the nucleotide sequence for the M2 protein.
  • the sequence encoding Meti to Lys 2S2 from the M sequence was used to express Ml protein in S2 cells. This sequence was derived from the nucleotide sequence for the H5N1 M sequence contained in accession number AF046090 (GenBank, www.ncbi.nlm.nih.gov).
  • the Ml protein is not one that is normally secreted from the cell, for this work the Ml protein, as defined above, was linked to the tPA secretion signal of the Drosophila expression plasmid to produce a secreted form of the truncated M protein.
  • the methods used to clone, transform, express and characterize the Ml protein are those described in Example 1. Upon selection of stable cell lines, the cells were screened for expression of the secreted form of the H5N1 Ml target protein. The expression of the described Ml subunit resulted in a uniform product of the expected molecular weight. The u i uuHwc has been estimated to be 15 to 20 ⁇ g/ml.
  • the column was washed with buffer A containing 15OmM NaCl until baseline absorbance was achieved. Bound material was eluted by a step gradient comprised of buffer A containing 0.5M and IM NaCl. Ml protein was eluted in the 0.5M NaCl step and was subsequently further purified by size exclusion chromatography on a Sephacryl S-100 column (1.5 x 94cm) using HmM phosphate buffered saline (14OmM NaCl), pH 7.2, as column buffer. The fractions containing Ml protein were pooled and concentrated by ultrafiltration for characterization.
  • H5 antigens expressed and purified according to the invention were evaluated in Balb/c mice.
  • H5 HA-A9-heads with or without H5N1 Ml protein were tested for immunogenic potential.
  • Groups of 5-9 female Balb/c mice aged 6-8 weeks were immunized by the subcutaneous route with the recombinant antigen(s) or appropriate . with GPI-0100 (250 ⁇ g/dose) as adjuvant in a total volume of 0.2 ml. Animals received 2 doses of vaccine at a 4 week interval. Seven days after the last dose of vaccine 4 mice/group were euthanized and spleens collected for analysis of cellular immune responses as described below.
  • ELISA assays Antibodies to the influenza proteins (H5 HA heads and H5 Ml proteins) were titrated by an ELISA technique, using a microplate format with wells coated with the specific antigen. Following coating, the wells were blocked with a serum or albumin containing buffer, and then standard ELISA steps were conducted with an alkaline- phosphatase or peroxidase conjugated secondary antibody.
  • HI assays were performed as described by standard methods (Kendal et al., CDC (1982) pB-17-B35) at Southern Research Institute (Frederick, MD).
  • Complement fixation assays Mouse sera were tested for complement fixation activity with the influenza antigens using a quantitative microcomplement fixation assay. Briefly, commercially obtained complement (guinea pig serum), hemolysin (rabbit anti-sheep erythrocyte stromata serum), and sheep erythrocytes (Cedarlane Laboratories, Hornby, Ontario, Canada) were used as the test indicator system and optimal concentrations for use determined by preliminary titrations (Lieberman, et al., Infect Immunol. (1979) 23:509-521). Dilutions of the purified antigens and mouse antisera were mixed and incubated with diluted complement in buffer on ice for 16 hrs.
  • Controls in which antigen or antiserum were omitted were included. Sheep erythrocytes sensitized by prior incubation with hemolysin were then added to the antigen+antiserum+complement mixture and incubated at 37°C for 60 min. The reaction mixtures were centrifuged and the absorbance of the supernatants at 413 nm o a ne ve e me uegree oi complement fixation by the antigen/antiserum combination, and the dilution of antiserum yielding 50% complement fixation can be determined. Thus, the complement fixing activities of different antisera to influenza antigens were directly compared.
  • Splenocyte preparations Splenectomies were performed 7 days post dose 2 on 4 mice each from groups 2 and 3. Splenocyte suspensions were prepared from each mouse spleen, erythrocytes lysed with NH 4 CI, and the final cell pellet washed and resuspended in cell culture medium. Cell counts were performed on each suspension using a Coulter counter, and suspensions diluted to 2 x ⁇ cells/ml with culture medium. Splenocytes from individual mice were cultured separately.
  • Lymphocyte proliferation assays Aliquots (0.1 ml) of each splenocyte suspension were dispensed into wells of a 96-well cell culture plate. The respective antigens were then added to the wells containing each of the cell suspensions (in quadruplicate) at a final concentration of 5 ⁇ g/ml (final volume of 0.2 ml/well). Wells with unstimulated (antigen omitted) cell suspensions were also included.
  • Cytokine production assays Aliquots (0.5 ml) of each splenocyte suspension were dispensed into wells of a 24- well cell culture plate. Five ⁇ g of the same antigens used for lymphocyte proliferation were then dispensed into the wells containing each of the cell suspensions (final volume of 1.0 ml/well). Unstimulated cell suspensions were tested as well as controls. Cultures were incubated for 4 days at 37°C/5% CO 2 / humidified. The culture supernatants were then harvested and frozen for analysis for specific cytokines. Cytokines in splenocyte culture supernatants were assayed using a flow cytometric bead array assay (BD Biosciences Pharmingen Corp., San Diego CA).
  • mice were capable of responding to stimulation with either antigen in vitro by proliferation and production of IFN- ⁇ and IL-5 (as well as TNF- ⁇ , IL-2, and IL-4; data not shown).
  • This cell- mediated immune response may be important in providing protective immunity against influenza to specific populations of subjects, such as elderly individuals (McElhaney JE, et al., J. Immunol. 176:6333-6339, 2006).
  • H5 HA subunit proteins specifically H5 HA-Ecto-mut and H5 HA-A9-head
  • H5 HA-Ecto-mut and H5 HA-A9-head were evaluated in Balb/c mice.
  • Groups of 5-10 female Balb/c mice aged 6-8 weeks were immunized by the intramuscular route with the recombinant p na ro s.
  • c n ere e vere as u a c ⁇ oi ntigen s wi or without alhydrogel (0.5 mg/dose) or GPI-0100 (250 ⁇ g/dose) as adjuvant in a total volume of 0.2 ml.
  • mice per group received 2 immunizations, the other five received 3 immunizations.
  • mice The immunogenicity of S2 expressed H3 HA-Ecto subunits with or without H5 Ml subunits was evaluated in Balb/c mice. Groups of 5-10 female Balb/c mice aged 6-8 weeks were immunized by the intramuscular route with the recombinant antigens or appropriate controls. Vaccines were delivered as a formulation of antigen(s) with or without alum (0.5 mg/dose) or GPI-0100 (250 ⁇ g/dose) as adjuvant in a total volume of 0.2 ml. Animals received 2 doses of vaccine at a 4 week interval or 3 doses of vaccine at a 3 week interval as indicated in Table 5 below. Two weeks after the last dose of vaccine, animals were y ELISA as described previously in Example 4. Results are shown in Figure 5.
  • H3 HA antigen is immunogenic.
  • the immunogenicity is increased when adjuvanted with alum or GPI-0100.
  • the addition of Ml to the immunizing vaccine did not significantly affect the titers to the HA antigen. No detectable antibody titers were raised in the adjuvant control groups (data not shown).
  • mice The immunogenicity of a dose range of S2 expressed H3 HA-Ecto or H3 HA-foldon subunits with or without Ml protein was evaluated in Balb/c mice. Groups of female Balb/c mice aged 6-8 weeks were immunized by the intramuscular route with the recombinant antigens or appropriate controls. Vaccines were delivered as a formulation of antigen(s) with or without alhydrogel (0.5 mg/dose) or GPI-0100 (250 ⁇ g/dose) as adjuvant in a total volume of 0.2 ml. Animals received 2 doses of vaccine at a 4 week interval or 3 doses of vaccine at a 3 week interval. Two weeks after the last dose of vaccine, animals were euthanized and serum samples tested for reactivity with recombinant proteins by ELISA as described previously in Example 5.
  • mice were immunized a minimum of twice; a maximum of three times, 28 days apart with 1 - 50 ⁇ g of H5 vaccine antigens (ectodomain, ectodomain + Ml or foldon). Two weeks after the final immunization, The mice were challeneged with a lethal dose of A/Vietnam /1203/04 in the following example. The mice were observed for morbidity and mortality for 14 days post infection. Lungs were taken from a subset of mice to determine viral titers using standard methods (Lu, et al., J. of Virol. (1999) 7:5903-5911).
  • Gregoriades, A The membrane protein of influenza virion extracted from virus and infected cells with acidic chloroform-methanol. Virology. 1973; 54:369-383
  • Heinen PP de Boer-Luijte EA, Bianchi AT. 2002. Respiratory and systemic humoral and cellular immune responses of pigs to a heterosubtype influenza A virus infection. J. Gen Virol. 82(Pt 11):2697-2707.
  • McElhaney JE Overcoming the challenges of immunosenescence in the prevention of acute respiratory illness in older people. Conn. Med. 2003 67:469-74 McElhaney JE, Xie D, Hager WD, Barry MB, Wang Y, Kleppinger A, Ewen C, Kane KP,
  • Pawelec G Immunosenescence and human longevity. Biogerontology 2003; 4:167-70 Pawelec G, Barnett Y, Fossey R, Frasca D, Globerson et al., (2002) Front. Biosci. 7:dl056- 183.
  • Influenza virus-like particles comprised of the HA, NA and Ml proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice.
  • Vanlandschoot P., E. Beirnaert, S. Neirynck, X. Saelens, W. Min Jou, and W. Fiers.
  • Wilson IA Skehel JJ, Wiley DC. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3A resolution. Nature 1981; 289:366-673.
  • Zhirnov OP Isolation of matrix protein Ml from influenza viruses by acid-dependent extraction with nonionic detergent. Virology 1992; 186:327-330.

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Abstract

L'invention concerne des protéines de la grippe, y compris des protéines sous-unitaires et des compositions immunogènes pouvant être utilisées, avec ou sans adjuvants, comme vaccins antigrippaux dans des modèles animaux et chez l'homme. Les protéines recombinées sont exprimées à partir de cellules d'insectes transformées qui contiennent des copies intégrées des cassettes d'expression appropriées dans leur génome. L'invention utilise un système d'expression de Drosophila melanogaster pour produire de grandes quantités de protéines sous-unitaires recombinées dont la conformation est analogue à celle de la protéine native.
PCT/US2006/032353 2005-08-16 2006-08-16 Vaccin a sous-unites recombinees du virus de la grippe WO2007022425A2 (fr)

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AU2006279323A AU2006279323B2 (en) 2005-08-16 2006-08-16 Influenza recombinant subunit vaccine

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WO2007085969A3 (fr) * 2006-01-27 2007-12-27 Novartis Vaccines & Diagnostic Vaccins contre la grippe contenant des hémaglutinines et des protéines matricielles
EP2147121A2 (fr) * 2007-04-26 2010-01-27 Hawaii Biotech, Inc. Vecteurs d'expression synthétiques pour cellules d'insecte
GB2471093A (en) * 2009-06-17 2010-12-22 Cilian Ag Viral protein expression in ciliates
US20110135676A1 (en) * 2008-06-12 2011-06-09 Khatijah Mohd Yusoff Novel antiviral peptide against avian influenza virus h9n2
WO2012011868A1 (fr) * 2010-07-23 2012-01-26 Osterhaus A D M E Vaccin antigrippal
US8420102B2 (en) 2006-03-07 2013-04-16 Vaxinnate Corporation Compositions that include hemagglutinin
US8932598B2 (en) 2012-08-28 2015-01-13 Vaxinnate Corporation Fusion proteins and methods of use
US8932605B2 (en) 2008-04-18 2015-01-13 Vaxinnate Corporation Deletion mutants of flagellin and methods of use
WO2015093996A1 (fr) * 2013-12-20 2015-06-25 Instytut Biochemii I Biofizyki Pan Antigène, vaccin contre la grippe, système pour la fabrication d'un vaccin, procédé de production d'un antigène et utilisation d'un antigène pour produire un vaccin contre la grippe
WO2015199564A1 (fr) * 2014-06-24 2015-12-30 Instytut Biotechnologii i Antybiotyków Protéine hémagglutinine du virus de la grippe en tant qu'antigène vaccinant

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WO2007085969A3 (fr) * 2006-01-27 2007-12-27 Novartis Vaccines & Diagnostic Vaccins contre la grippe contenant des hémaglutinines et des protéines matricielles
EP3753574A1 (fr) * 2006-01-27 2020-12-23 Seqirus UK Limited Vaccins contre la grippe contenant de l'hémagglutinine et des protéines de matrice
EA015271B1 (ru) * 2006-01-27 2011-06-30 Новартис Вэксинс Энд Диагностикс Гмбх & Ко Кг Противогриппозные вакцины, содержащие гемагглютинин и белки матрикса
US9200042B2 (en) 2006-03-07 2015-12-01 Vaxinnate Corporation Flagellin fusion proteins
US9056901B2 (en) 2006-03-07 2015-06-16 Vaxinnate Corporation Methods of making hemagglutinin proteins
US8945579B2 (en) 2006-03-07 2015-02-03 Vaxinnate Corporation Methods of treatment with compositions that include hemagglutinin
US8420102B2 (en) 2006-03-07 2013-04-16 Vaxinnate Corporation Compositions that include hemagglutinin
US8198079B2 (en) 2007-04-26 2012-06-12 Merck Sharp & Dohme Corp. Synthetic expression vectors for insect cells
EP2147121A4 (fr) * 2007-04-26 2011-09-14 Merck Sharp & Dohme Vecteurs d'expression synthétiques pour cellules d'insecte
EP2147121A2 (fr) * 2007-04-26 2010-01-27 Hawaii Biotech, Inc. Vecteurs d'expression synthétiques pour cellules d'insecte
US9205138B2 (en) 2008-04-18 2015-12-08 Vaxinnate Corporation Deletion mutants of flagellin and methods of use
US8932605B2 (en) 2008-04-18 2015-01-13 Vaxinnate Corporation Deletion mutants of flagellin and methods of use
US9211320B2 (en) 2008-04-18 2015-12-15 Vaxinnate Corporation Deletion mutants of flagellin and methods of use
US20110135676A1 (en) * 2008-06-12 2011-06-09 Khatijah Mohd Yusoff Novel antiviral peptide against avian influenza virus h9n2
US8883480B2 (en) * 2008-06-12 2014-11-11 Universiti Putra Malaysia Antiviral peptide against avian influenza virus H9N2
GB2471093A (en) * 2009-06-17 2010-12-22 Cilian Ag Viral protein expression in ciliates
US8377682B2 (en) 2009-06-17 2013-02-19 Sanofi Pasteur S.A. System for the heterologous expression of a viral protein in a ciliate host cell
CN107281478A (zh) * 2010-07-23 2017-10-24 诺瓦瓦克斯公司 流感疫苗
RU2583297C2 (ru) * 2010-07-23 2016-05-10 Исконова Аб Противогриппозная вакцина
JP2016190846A (ja) * 2010-07-23 2016-11-10 イスコノバ アーベー インフルエンザワクチン
CN103052401A (zh) * 2010-07-23 2013-04-17 伊斯克诺瓦公司 流感疫苗
US10485863B2 (en) 2010-07-23 2019-11-26 Novavax AB Influenza vaccine
US10736958B2 (en) 2010-07-23 2020-08-11 Novavax AB Influenza vaccine
WO2012011868A1 (fr) * 2010-07-23 2012-01-26 Osterhaus A D M E Vaccin antigrippal
US8932598B2 (en) 2012-08-28 2015-01-13 Vaxinnate Corporation Fusion proteins and methods of use
WO2015093996A1 (fr) * 2013-12-20 2015-06-25 Instytut Biochemii I Biofizyki Pan Antigène, vaccin contre la grippe, système pour la fabrication d'un vaccin, procédé de production d'un antigène et utilisation d'un antigène pour produire un vaccin contre la grippe
WO2015199564A1 (fr) * 2014-06-24 2015-12-30 Instytut Biotechnologii i Antybiotyków Protéine hémagglutinine du virus de la grippe en tant qu'antigène vaccinant
US10398770B2 (en) 2014-06-24 2019-09-03 Instytut Biotechnologii I Anty-Biotykow Influenza virus hemagglutinin protein as a vaccine antigen

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US20070042002A1 (en) 2007-02-22
AU2006279323B2 (en) 2013-08-01
EP1945250A4 (fr) 2010-05-19

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